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Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...

Ehime University

Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice ( ) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from  mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including , which is reported to be an IL1-related gene and to affect chondrocyte differentiation.  knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as  These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation.

Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression.

Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor that is involved in osteoclast differentiation in our previous report. However, the biological functions of Zscan10 are not fully understood except its roles in the maintenance of genome stability and pluripotency of embryonic stem cells. Therefore, the purpose of this study was to clarify the function of Zscan10 in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established by genome editing using CRISPR/Cas9 and single cell sorting. Then, control (Ctrl) and KO cells were differentiated into osteoclasts by RANKL stimulation. We observed that TRAP activity and the expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells compared to Ctrl cells. These results suggest that Zscan10 might regulate transcription of the genes that negatively control osteoclastogenesis. To understand gene expression profiles controlled by Zscan10, RNA-seq was performed and stringent analyses identified the haptoglobin gene (Hp) as a possible target of Zscan10. In addition, ChIP against Zscan10 revealed that Zscan10 could interact with its binding motif located near the Hp gene locus as well as the transcription start site of Hp, suggesting that Zscan10 can directly regulate transcription of Hp. Finally, to examine the effects of Hp on osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). rHp treatment suppressed TRAP activity of KO cells without affecting cell viability. Furthermore, it has been reported that Hp KO mice exhibit decreased bone mass and increased osteoclast number. Importantly, hemolytic disease patients exhibited decreased serum level of Hp as well as low bone mineral density. Taken together, this study suggests that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.

Zscan10 suppresses osteoclast differentiation by regulating expression of Haptoglobin.

The prognosis of patients with progressive prostate cancers that are hormone refractory and/or have bone metastasis is poor. Multiple therapeutic targets to improve prostate cancer patient survival have been investigated, including orphan GPCRs. In our study, we identified G Protein-Coupled Receptor Class C Group 5 Member A (GPRC5A) as a candidate therapeutic molecule using integrative gene expression analyses of registered data sets for prostate cancer cell lines. Kaplan-Meier analysis of TCGA data sets revealed that patients who have high GPRC5A expression had significantly shorter overall survival. PC3 prostate cancer cells with CRISPR/Cas9-mediated GPRC5A knockout exhibited significantly reduced cell proliferation both in vitro and in vivo. RNA-seq revealed that GPRC5A KO PC3 cells had dysregulated expression of cell cycle-related genes, leading to cell cycle arrest at the G2/M phase. Furthermore, the registered gene expression profile data set showed that the expression level of GPRC5A in original lesions of prostate cancer patients with bone metastasis was higher than that without bone metastasis. In fact, GPRC5A KO PC3 cells failed to establish bone metastasis in xenograft mice models. In addition, our clinical study revealed that GPRC5A expression levels in prostate cancer patient samples were significantly correlated with bone metastasis as well as the patient's Gleason score (GS). Combined assessment with the immunoreactivity of GPRC5A and GS displayed higher specificity for predicting the occurrence of bone metastasis. Together, our findings indicate that GPRC5A can be a possible therapeutic target and prognostic marker molecule for progressive prostate cancer.

GPRC5A facilitates cell proliferation through cell cycle regulation and correlates with bone metastasis in prostate cancer.

The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-κB pathway. Missense mutations in the  gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free-based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine  knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-κB signaling. Of note, arthritic inflammation was suppressed in -deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-κB signaling via tumor necrosis factor alpha (TNFα) stimulation and the linear ubiquitination assembly complex (LUBAC). -knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLD variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.

The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD.

Macrophages adapt to microenvironments, and change metabolic status and functions to regulate inflammation and/or maintain homeostasis. In joint cavities, synovial macrophages (SM) and synovial fibroblasts (SF) maintain homeostasis. However, under inflammatory conditions such as rheumatoid arthritis (RA), crosstalk between SM and SF remains largely unclear.

Reprogramming of synovial macrophage metabolism by synovial fibroblasts under inflammatory conditions.

Epigenetic transcriptional regulation is essential for the differentiation of various types of cells, including skeletal muscle cells. DNA methyltransferase 1 (Dnmt1) is responsible for maintenance of DNA methylation patterns via cell division. Here, we investigated the relationship between Dnmt1 and skeletal muscle regeneration. We found that Dnmt1 is upregulated in muscles during regeneration. To assess the role of Dnmt1 in satellite cells during regeneration, we performed conditional knockout (cKO) of Dnmt1 specifically in skeletal muscle satellite cells using Pax7 mice and Dnmt1 flox mice. Muscle weight and the cross-sectional area after injury were significantly lower in Dnmt1 cKO mice than in control mice. RNA sequencing analysis revealed upregulation of genes involved in cell adhesion and apoptosis in satellite cells from cKO mice. Moreover, satellite cells cultured from cKO mice exhibited a reduced number of cells. These results suggest that Dnmt1 is an essential factor for muscle regeneration and is involved in positive regulation of satellite cell number.

DNA maintenance methylation enzyme Dnmt1 in satellite cells is essential for muscle regeneration.

Androgens have a robust effect on skeletal muscles to increase muscle mass and strength. The molecular mechanism of androgen/androgen receptor (AR) action on muscle strength is still not well known, especially for the regulation of sarcomeric genes. In this study, we generated androgen-induced hypertrophic model mice, myofiber-specific androgen receptor knockout (cARKO) mice supplemented with dihydrotestosterone (DHT). DHT treatment increased grip strength in control mice but not in cARKO mice. Transcriptome analysis by RNA-seq, using skeletal muscles obtained from control and cARKO mice treated with or without DHT, identified a fast-type muscle-specific novel splicing variant of  as a target of AR in skeletal muscles.  knockout mice exhibited decreased maximum isometric torque of plantar flexion and passive stiffness of myofibers due to reduced phosphorylation of Myomesin 1 protein. This study suggests that androgen-induced skeletal muscle strength is mediated with Mylk4 and Myomesin 1 axis.

Myofiber androgen receptor increases muscle strength mediated by a skeletal muscle splicing variant of Mylk4.

Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation with aberrant epigenetic alterations, eventually leading to joint destruction. However, the epigenetic regulatory mechanisms underlying RA pathogenesis remain largely unknown. Here, we showed that ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) is a central epigenetic regulator that orchestrates multiple pathogeneses in RA in a suppressive manner. UHRF1 expression was remarkably upregulated in synovial fibroblasts (SFs) from arthritis model mice and patients with RA. Mice with SF-specific Uhrf1 conditional knockout showed more severe arthritic phenotypes than littermate controls. Uhrf1-deficient SFs also exhibited enhanced apoptosis resistance and upregulated expression of several cytokines, including Ccl20. In patients with RA, DAS28, CRP, and Th17 accumulation and apoptosis resistance were negatively correlated with UHRF1 expression in synovium. Finally, Ryuvidine administration stabilized UHRF1 ameliorated arthritis pathogeneses in a mouse model of RA. This study demonstrated that UHRF1 expressed in RA SFs can contribute to negative feedback mechanisms that suppress multiple pathogenic events in arthritis, suggesting that targeting UHRF1 could be one of the therapeutic strategies for RA.

Article	Year
Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression. Development (Cambridge, England)\| PubMed ID: 29180567	2018
Zscan10 suppresses osteoclast differentiation by regulating expression of Haptoglobin. Bone\| PubMed ID: 30771488	2019
GPRC5A facilitates cell proliferation through cell cycle regulation and correlates with bone metastasis in prostate cancer. International journal of cancer\| PubMed ID: 31276604	2020
The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD. The Journal of biological chemistry\| PubMed ID: 31366726	2019
Reprogramming of synovial macrophage metabolism by synovial fibroblasts under inflammatory conditions. Cell communication and signaling : CCS\| PubMed ID: 33256735	2020
DNA maintenance methylation enzyme Dnmt1 in satellite cells is essential for muscle regeneration. Biochemical and biophysical research communications\| PubMed ID: 33310192	2021
Myofiber androgen receptor increases muscle strength mediated by a skeletal muscle splicing variant of Mylk4. iScience\| PubMed ID: 33870126	2021
Epigenetic regulator UHRF1 orchestrates proinflammatory gene expression in rheumatoid arthritis in a suppressive manner. The Journal of clinical investigation\| PubMed ID: 35472067	2022

Noritaka Saeki

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